Interaction of Pyrene-3-sulfonate with Lipid Monolayers - American

DOMA monolayers which were transferred from the air/water interface to the mercury surface. .... Baker Chemicals, Holland, and Kemika, Croatia, respec...
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Langmuir 1996, 12, 5387-5392

5387

Interaction of Pyrene-3-sulfonate with Lipid Monolayers Z. Kozarac* and B. C Ä osovic´ Rud i er Bosˇ kovic´ Institute, Center for Marine Research-Zagreb, P.O. Box 1016, HR-10001 Zagreb, Croatia

R. C. Ahuja, D. Mo¨bius, and W. Budach Max-Planck-Institute fu¨ r Biophysikalische Chemie, Postfach 2841, D-37018 Go¨ ttingen, Germany Received October 18, 1995. In Final Form: July 3, 1996X Interaction of sodium pyrene-3-sulfonate (NaPyS) with different lipid monolayers has been studied by fluorescence and reflection spectroscopy, surface pressure measurements, and electrochemical methods. NaPyS interacts with dioctadecyldimethylammonium bromide (DOMA) monolayers at the air/water interface due to the Coulombic interaction with the positively charged head groups, while no interaction with dipalmitoylphosphatidylcholine (DPPC) monolayers has been noticed. NaPyS from solution also influenced DOMA monolayers which were transferred from the air/water interface to the mercury surface. The enhancement of the transport of cadmium ions across a DOMA film modified by NaPyS adsorption was observed.

1. Introduction Natural aquatic systems contain a large number of organic substances with different functional groups and different hydrophobic properties. Adsorption at natural interfaces (air/water, water/particles, biota/water, and water/sediment) is determined by hydrophobic and/or electrostatic interactions of the adsorbate and the interface. Electrostatic interactions of hydrophilic ions or molecules from the bulk solution with functional groups of adsorbed hydrophobic substances, which are found to be predominant in organic matter adsorbed at model and natural phase boundaries,1 may be responsible for the surface excess of these solute species at the natural interfaces. Polycyclic aromatic hydrocarbons (PAHs) represent highly dangerous pollutants in the environment and are included on the list of priority pollutants of the Environmental Protection Agency.2 Studies of the adsorption behavior of PAHs and their salts, as well as their interactions with lipids, which represent constituents of biological membranes and substances naturally occurring at different phase boundaries, might be very helpful in evaluating the processes that influence environmental behavior and effects of organic contaminants in natural aquatic systems. In order to get better insight into the complex phenomena of the adsorption of pyrene and pyrene derivatives at phase boundaries, we have started a systematic physicochemical investigation of these compounds at the monolayer/subphase interface. Recently we reported on the studies of complex pyrene-3-sulfonate/dioctadecyldimethylammonium bromide (PyS-/DOMA) monolayers, prepared by a cospreading technique at the air/water interface. It was found that a densely packed monolayer of PyS- is formed under the head groups of the anchor lipid monolayer and that pyrene chromophores are inclined with respect to the air/water interface. The influence of the subphase composition on the molecular organization X

Abstract published in Advance ACS Abstracts, October 1, 1996.

(1) C Ä osovic´, B.; Vojvodic´, V. Mar. Chem. 1989, 28, 183. (2) Keith, H. L.; Telliard, W. A. Environ. Sci. Technol. 1979, 13, 416.

S0743-7463(95)00908-5 CCC: $12.00

of PYS-/DOMA cospread monolayers at the air/water interface was also investigated.3 Here we report on the studies of the interaction of sodium pyrene-3-sulfonate (NaPyS) with different lipid monolayers which were performed by surface pressurearea measurements, fluorescence and reflection spectroscopy, and electrochemical methods. 2. Experimental Section 2.1. Monolayer Studies at the Air/Water Interface. Surface pressure-area isotherms have been measured on a rectangular thermostated Teflon trough enclosed in a tight box. For measurement of surface pressure, a Wilhelmy balance (20 mm wide filter paper) was used. Fluorescence measurements were performed by using a PerkinElmer Luminescence spectrometer LS-5, modified for in situ measurements at the air/water interface.4 The excitation wavelength was 348 nm, and the emission spectrum was measured in the range between 370 and 550 nm. The time dependence of excimer fluorescence was measured at the wavelength 475 nm. Reflection spectroscopic measurements have been performed with a reflection spectrometer for measurement under a normal incidence of light5 using a Fromherz round trough.6 The reflection was measured and expressed as the difference in the reflectivity between the monolayer free solution surface and the surface covered with a monolayer. Monolayers were prepared as follows. NaPyS solution was put in the trough, and the lipid monolayers were spread on top of the solution and compressed to the desired initial pressure. Since the trough was not deep enough to ensure efficient mixing and homogeneous distribution of NaPyS in the solution without breaking the monolayer, the injection of NaPyS underneath the previously formed lipid monolayers was not a convenient method. 2.2. Electrochemical Measurements. Electrochemical studies have been performed by using phase sensitive alternating current voltammetry.7 By using this method, the capacity current reflecting the properties of the electrode double layer at the (3) Kozarac, Z.; Ahuja, R. C.; Mo¨bius, D. Langmuir 1995, 11, 568. (4) Budach, W. Elektrostatische, dynamische und optische Eigenschaften von organisierte Monofilmen hinsichtlich des Aufbaus von Sensor-Schichtsystemen. Ph.D. Thesis, Go¨ttingen, 1991. (5) Gru¨niger, H.; Mo¨bius, D.; Meyer, H. J. Chem. Phys. 1983, 79, 3701. (6) Fromherz, P. Rev. Sci. Instrum. 1975, 46, 1380. (7) Smith, D. E.; A.c. polarography and related techniques: Theory and practice. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1966; Vol. 1.

© 1996 American Chemical Society

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Kozarac et al. Deionized water from a Mili-Q system (Millipore Corp.) was used for preparing the subphase in monolayer studies and all solutions in electrochemical experiments. Chloroform (HPLC) and hexane (p.a.a. grade) have been used as spreading solvents for monolayers and were obtained from Baker Chemicals, Holland, and Kemika, Croatia, respectively.

3. Results and Discussion

Figure 1. Chemical structures of NaPyS, DOMA, and DPPC. electrode/solution interface and/or the Faradaic current, resulting from possible redox processes at the electrode, can be measured selectively. Lipid films were prepared by spreading from hexane solutions onto the electrolyte solutions with and without NaPyS, respectively, and were transferred to the mercury electrode by vertical dipping of the electrode through the film, as described elsewhere in more detail.8-10 The interaction of NaPyS with lipid films transferred to the mercury surface was determined by capacity measurements (outof-phase signal measurements at a frequency of 75 Hz) with prior accumulation of surfactant at the selected convenient potential (-0.6 V vs Ag/AgCl) during 60 s. The influence of the adsorbed layer on the oxido-reduction processes of cadmium was followed by measuring Faradaic current (in-phase signal measurements) after accumulation of NaPyS at -0.4 vs Ag/AgCl. All measurements were performed with a Methrom E 506 polarograph. A standard polarographic Methrom cell of 100 cm3 equipped with a three-electrode system was used. A hanging mercury drop electrode (HMDE, Methrom, Switzerland) was used as the working electrode, an Ag/AgCl electrode was the reference electrode, and a platinum wire was the auxiliary electrode. Pure nitrogen was used for deaeration of the solution in the Faradaic current measurements. All measurements were made at room temperature. 2.3. Chemicals. Sodium pyrene-3-sulfonate (NaPyS) was obtained from Molecular Probes. Dioctadecyldimethylammonium bromide (DOMA), dipalmitoylphosphatidylcholine (DPPC), and egg lecithin were purchased from Sigma Chemical Co. and used as received. Structural drawing of the compounds are presented in Figure 1. Sodium chloride and cadmium chloride were from Merck. Sodium chloride, used as supporting electrolyte in electrochemical measurements, was heated at 723 K for several hours to eliminate traces of organic matter. A saturated solution of NaCl was additionally purified with charcoal. (8) Miller, I. R.; Rishpon, J.; Nenenbaum, A. Bioelectrochem. Bioenerg. 1976, 3, 528. (9) Nelson, A. Anal. Chim. Acta 1987, 194, 139. (10) Kozarac, Z.; Klaric´, R.; Dragcˇevic´, D Ä osovic´, B. Colloids Surf. i .; C 1991, 56, 279.

3.1. Surface Pressure Measurements. Measurements of the surface pressure of NaPyS solutions in a time period of 2 h did not show any considerable change in surface pressure, even at rather high concentrations of 100 µM NaPyS (∆π ) 3.7 mN/m), indicating no appreciable accumulation of NaPyS on the free air/solution interface. If lipid monolayers are present on top of the solution, both electrostatic and hydrophobic interactions between the lipid and solute molecules can take place, thus influencing the surface characteristics like surface tension and surface potential. As a result of the head-group charge of the monolayer, charged solute or counterions are attracted to the interface, so that the interfacial ion concentration is significantly higher than that in the bulk. The concentrations of counterions at or close to the interface are dependent on the nature (size, valence, polarizability, hydration) and concentrations of the ions, the pH of the subphase, and the surface charge density of the monolayers. The adsorption of counterions at the monolayer/subphase interface leads to significant changes in intermolecular electrostatic interactions at the interface. For the studies of the interaction of NaPyS with lipid monolayers, the zwitterionic DPPC and the positively charged DOMA have been chosen. DPPC belongs to the group of phospholipids (lecithins), which constitute the major lipid material in biomembranes. The structure and ion permeability of both synthetic and natural lecithin are probably the most extensively used models in the studies of phenomena existing in biomembranes.11-13 Although DPPC is not a perfect model for a cell membrane, it is very often used in monolayer studies, since it is insoluble in water and forms a very stable monolayer at the air/water interface compared to egg lecithin. DOMA is a cationic amphiphilic lipid which forms well defined and stable monolayers at the air/water interface. The morphology and molecular organization characteristics of DOMA monolayers have been extensively studied, and a strong dependence on the nature and concentrations of counterions in the subphase was found.14-16 The surface pressure-area (π-A) isotherms of a DPPC monolayer spread on water (curve 1) and on a subphase containing 2 µM NaPyS (curve 2) are shown in Figure 2. The isotherms have been measured 2 h after spreading the monolayer. DPPC spread on water exhibits a rather condensed film with a phase transition around 5 mN/m (curve 1). The isotherm of the DPPC monolayer shows only small differences when measured on the NaPyScontaining subphase, even 2 h after spreading. It seems to be in contradiction with the penetration of other PAHs in phospholipid monolayers as was reported by Cadenhead13 for β-naphtol interacting with DPPC and (11) Miller, I. R.; Bach, D. J. Colloid Interface Sci. 1969, 29, 250. (12) Mu¨ller, E.; Do¨rfler, H. D. Bioelectrochem. Bioenerg. 1980, 7, 459. (13) Cadenhead, D. A. Monomolecular films as biomembrane models. In Structure and Properties of Cell Membranes; Benga, G. M. D., Ed.; CRC Press, Inc.: Boca Raton, FL, 1985; Vol. 3. (14) Goddard, E. D.; Kao, O.; Kung, H. C. J. Colloid Interface Sci. 1968, 27, 616. (15) Marra, J. J. Phys. Chem. 1986, 90, 2145. (16) Ahuja, R. C.; Caruso, P.-L.; Mo¨bius, D. Thin Solid Films 1994, 242, 195.

Interaction of Pyrene-3-sulfonate with Lipid Monolayers

Figure 2. Surface pressure-area (π-A) isotherm of the DPPC monolayer on a water subphase (curve 1) and on a subphase containing 2 µM NaPyS (curve 2).

Figure 3. Surface pressure-area (π-A) isotherms of the DOMA monolayer on a water subphase (curve 1) and on a subphase containing 2 µM NaPyS (curve 2).

by us for DMPC monolayers penetrated by pyrene.17 In the case of β-naphtol penetration to the DPPC monolayer, it is obvious that the concentrations of solute are much higher than in our case. The concentration of NaPyS in solution was 2 × 10-6 M, and either β-naphtol did not interact with DPPC in this concentration range. The concentration of pyrene in cospread monolayers with DMPC was even higher (5 × 10-4 M), which can be an explanation for the observed difference in the interaction of β-naphtol and pyrene with phosphatidylcholines compared to NaPyS. However, in the same low concentration range NaPyS showed considerable influence on the DOMA monolayers, indicating the predominant role of electrostatic interactions. The surface pressure-area (π-A) isotherms of a DOMA monolayer on water (curve 1) and on an aqueous 2 µM NaPyS solution (curve 2) are presented in Figure 3. The isotherms have been measured 2 h after spreading the monolayer. The DOMA monolayer spread on water exhibits an isotherm of the liquid-expanded type which changes to a liquid-condensed isotherm as the area/ molecule is reduced. The isotherm of the DOMA monolayer spread on the subphase containing 2 µM NaPyS shows the contraction with respect ot DOMA at larger areas, A g 0.8 nm2 (16 mN/m). Further compression until A ) 0.7 nm2 leads to a strong increase in surface pressure. The area/DOMA molecule for the monolayer spread on (17) Kozarac, Z.; C Ä osovic´, B.; Budach, W.; Mo¨bius, D. Croat. Chim. Acta, in press.

Langmuir, Vol. 12, No. 22, 1996 5389

NaPyS solution in the tightly packed state is larger than that for the DOMA monolayer spread on water. The condensation of the DOMA monolayer isotherm at small surface pressures can be attributed to the decreasing of the repulsive forces between the head groups of DOMA due to the neutralization of positive charge via electrostatic interaction with PyS- from solution. At higher surface pressures, an expansion of the DOMA monolayer spread on the NaPyS subphase with respect to the DOMA spread on water is observed. The phase transition of the DOMA monolayers spread on the NaPyS solution disappears too. The expansion of the π-A isotherms as a result of the lipid/solute interaction has usually been interpreted in terms of penetration or incorporation of solute molecules in the monolayer.18-20 However, an expansion of the area can also be observed when electrostatically adsorbed molecules are located underneath the lipid monolayer, as has already been shown.3,21,22 The analysis of the π-A isotherms of cospread DOMA/ PyS- monolayers in terms of different pyrene conformations showed that PyS- molecules cannot be incorporated in the hydrophobic part of the monolayer.3 The area per DOMA molecule in the condensed state in the complex monolayer at 40 mN/m was 0.75 nm2, which is more expanded than the expected value of 0.4 nm2 for neutral double-chained lipids. The measured area value of 0.75 nm2 is also less than the value of ca. 1 nm2 required if the pyrene molecules lie flat at the air/water interface. The only possible explanation is that pyrene molecules are located underneath the DOMA monolayer. The hypothesis that PyS- molecules are organized in the form of a submonolayer under the positively charged head groups of the densely packed DOMA monolayers was confirmed by reflection spectroscopic measurements of the reflection with linearly polarized light under oblique incidence.3 3.2. Fluorescence Measurements. Fluorescence measurements are very often used in studies of the adsorption of unsubstituted and substituted pyrenes. Pyrene has a relatively long excited state lifetime, a high quantum yield of fluorescence, and the ability of forming excimers, i.e. molecular complexes in the excited state.23,24 Figure 4 shows the emission spectra (range 370-550 nm) of subphases containing 2 µM NaPyS covered with DOMA and DPPC monolayers at 30 mN/m, respectively. The emission spectra are dominated by the monomer fluorescence arising from the excess NaPyS in the subphase, and emission bands were found at 378, 388, 396, 418, and 443 nm. For the DOMA monolayer we have found an additional excimer band located at 475 nm. For the DPPC monolayer no additional excimer band was observed. Excimer formation requires direct contact between PyS- chromophores. Even if the average distance of the adsorbed PyS- chromophores is large, as in the case of chromophore adsorption from the subphase, weak excimer emission can be expected due to the high mobility of the PyS- chromophores. Much higher excimer emission was found in mixed DOMA/PyS- films formed by the cospreading technique.3 It is interesting to compare the excimer emission of complex monolayers of DOMA/PySobtained through the techniques of adsorption and cospreading. In the cospreading technique, a large fraction (18) Verger, R.; Pattus, F. Chem. Phys. Lipids 1982, 30, 189. (19) Ter-Minassian Saraga, L. Langmuir 1985, 1, 391. (20) Ivanova, M. G.; Verger, R.; Bois, A. G.; Panaiotov, I. Colloids Surf. 1991, 54, 279. (21) Mo¨bius, D.; Gru¨nniger, H. Bioelectrochem. Bioenerg. 1984, 12, 375. (22) Ahuja, R. C.; Caruso, P. L.; Mo¨bius, D.; Wildburg, G.; Ringsdorf, H.; Philp, D.; Preece, J. A.; Stoddart, J. F. Langmuir 1993, 9, 1534. (23) Stevens, B.; Hutton, E. Nature 1960, 186, 1045. (24) Fo¨rster, Th. Angew. Chem. 1969, 81, 364.

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Figure 4. Excitation (range 250-370 nm, dashed line) and emission spectra (range 370-550 nm, full line) of the DOMA monolayers and emission spectrum (dotted line) of the DPPC monolayers on the subphase containing 2 µM NaPyS.

Figure 5. Time dependence of the excimer fluorescence (475 nm) of DOMA monolayers on a subphase containing 0.4, 1, and 2 µM NaPyS and of DPPC monolayers on a subphase containing 2 µM NaPyS.

of the PyS- is trapped at the interface immediately after spreading, resulting in higher local concentrations of PySand in a higher excimer emission. In the adsorption technique, the adsorption time for obtaining a complex ligand-lipid monolayer is much longer and dependent on the concentrations of PyS- in the subphase. In addition, we have also shown the excitation spectrum (range 250-370 nm) of the NaPyS subphase covered with a DOMA monolayer at 30 mN/m. Excitation bands are at 260, 265, 275, 330, and 344 nm. The results presented in Figure 5 show the excimer fluorescence at 475 nm versus time. Fluorescence was recorded from DOMA monolayers spread on subphases containing 0.4, 1, and 2 µM NaPyS and compressed to a surface pressure of 30 mN/m. The fluorescence at the same wavelength from a DPPC monolayer spread on 2 µM NaPyS and compressed to 30 mN/m is given as reference. The excitation was at the wavelength of 348 nm. It can be seen that the excimer fluorescence intensity depends on the concentration of NaPyS in the subphase and on time. The maximum is reached after approximately 100 min (saturation). A further increase of the NaPyS concentration in the subphase does not yield a significant increase of the excimer fluorescence. Therefore, we assume saturation for an approximately 100 min adsorption time and an approximately 2 µM subphase concentration of NaPyS. However, monomer emission can

Kozarac et al.

Figure 6. Reflection spectra of the DPPC (curve 1) and DOMA (curve 2) monolayers under normal incidence of light. Subphase: 3 µM NaPyS. Surface pressure: 30 mN/m.

be always expected, since pyrene chromophores present both at the interface and in solution contribute to the measured fluorescence. 3.3. Reflection Measurements. Reflection spectroscopy is based on the enhanced reflection due to the presence of chromophores in a monolayer at an air/water interface, without any contribution of the chromophores from the bulk solution.5 Therefore, reflection measurements provide better insight in the processes at the interface and have been performed to confirm the case in which PySis located at the interface. Reflection spectra for DPPC and DOMA monolayers spread on a 3 µM solution of NaPyS and compressed to 30 mN/m are shown in Figure 6. An enhanced reflection signal was obtained from the DOMA monolayer spread on the NaPyS solution, confirming the presence of pyrene chromophores at the interface. No signal was observed when the DPPC monolayer was spread on the NaPyS subphase, meaning that no interaction at the interface occurred. The same (i.e. no reflection signal) was observed when monolayers were spread on pure water. The studies of adsorption kientics at the monolayer/ subphase interface can provide a better understanding of the different parameters which determine the properties of the adsorbed monolayer. The kinetics of adsorption was measured as follows. The trough was filled with the subphase containing NaPyS in the concentration range from 0.03 to 3.0 µM. DOMA monolayers were spread on these subphases and compressed quickly to 30 mN/m. Subsequently the surface reflection ∆R (at 346 nm) was monitored at constant surface area for a period of 2 h. The results of the adsorption kinetics measurements are shown in Figure 7a. The change in ∆R reflects only the changes in the PyS- surface density and/or the orientation at the interface, without the contribution of the chromophores from the solution. The contribution of the DOMA monolayer to ∆R can be seen in a curve obtained on pure water (curve 1), which is identical with the reflection of the DOMA monolayers spread on 0.03 µM NaPyS. In the presence of NaPyS in the subphase, the reflection ∆R increases with time, reaching the saturation value (∆R ) 0.132%), for higher concentrations of NaPyS, within 2 h. The experiments were carried out with the precaution to minimize the time between the spreading of the monolayer and the beginning of the reflection measurements. Similar experiments with DPPC monolayers as a control have been performed, and no reflection kinetics was observed. The surface density σ of the adsorbate NaPyS is related to ∆R through a constant β, which is a property of the adsorbate but also depends on the orientation of the

Interaction of Pyrene-3-sulfonate with Lipid Monolayers

Langmuir, Vol. 12, No. 22, 1996 5391 Table 1. Surface Concentration σ of Adsorbate Calculated from Eq 3 for Different Concentrations of NaPyS c (µM)

t (min)

0.8 1.6 3.0

151.3 30.8 11.6

σ (10-6 mol/m2) 1.67 1.51 1.73 σavg ) 1.64 × 10-6

the diffusion coefficient D can be calculated from the Stokes-Einstein equation, according to which

D ) kT/6πηa

Figure 7. (a) Time dependence of the surface reflection (346 nm) of the DOMA monolayers spread on a subphase containing (1) 0.03, (2) 0.2, (3) 0.4, (4) 0.8, (5) 1.6, and (6) 3.0 µM NaPyS. Curve 1 also denotes the reflection of the DOMA monolayer spread on water. (b) Reflection vs the square root of time for the adsorption of PyS- at DOMA monolayers. Subphase concentration: (1) 0.8, (2) 1.6, and (3) 3 µM PyS-.

adsorbate at the interface. From the molecular area of NaPyS, which is ca. 1 nm2, if the pyrene molecules lie flat at the air/water interface, the surface density of a compact layer of adsorbate is given by σ ) 1/A. Thus the value σ ) 1.66 × 10-6 mol/m2 is obtained with the corresponding ∆R ) 1.32 × 10-3, which in turn (β ) σ/∆R) gives β ) 1.258 × 10-3 m2/mol. The time dependence of ∆R showed a linear relationship with the square root of time (Figure 7b), which is a good test of diffusion-controlled kinetics.25,26 The number of adsorbate molecules reaching the interface per unit time and unit area can be obtained by known relations of Fick’s law

dσ/dt ) D ∂c/∂x

(1)

dσ/dt ) Dc(Dtπ)-1/2

(2)

which after integration and expressing the concentration c in moles per liter gives

σ ) (11.3 × 10-4)D1/2ct1/2 (mol/cm2)

(3)

The σ value of the compact layer of adsorbate can be determined by introducing the t value obtained at the moment of attaining the saturation value of ∆R for the given concentrations of NaPyS. An approximate value of (25) Jehring, H. Elektrosorptionsanalyse mit der Wechselstrompolarographie; Akademie-Verlag: Berlin, 1974. (26) Bockris, J. O. M.; Reddy, A. K. Modern Electrochemistry, 3rd ed.; Plenum/Roseta: New York, 1977; Vol. 2, p 1056.

(4)

where k is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the subphase, and a is the radius of the molecule. Taking that η ) 1 cp for water at 298 K and a ≈ 0.564 nm, the value D ) 3.80 × 10-6 cm2/s is obtained. Using eq 3 the σ value was calculated for different concentrations of NaPyS, as presented in Table 1. The obtained average of σ ) 1.64 × 10-6 mol/m2 is in good agreement with the compact layer of NaPyS calculated above from the molecular area of NaPyS. 3.4. Electrochemical Measurements. Interactions of lipids with various species from the bulk solution can also be studied electrochemically by using lipid-coated mercury electrodes. The technique of transferring the lipid film from the air/water interface to the mercury surface was specially designed for studies of the lipidprotein interactions.8,27 and for investigations of the ion transport across natural membranes.28 Phospholipidcoated mercury electrodes represent also a very sophisticated system for the study of the structure and functioning of biological membranes29-31 as well as for the investigations of the aquatic chemistry of organic and inorganic micropollutants.9,32,33 The influence of NaPyS adsorbed from the solution on the DOMA monolayers transferred to the mercury electrode has been studied by phase sensitive ac voltammetry. In the potential region from -0.6 V toward more negative potentials, DOMA monolayers (surface concentrations below that of the condensed monolayer) transferred to the mercury surface show a capacitance minimum and two capacitance peaks at approximately -1.0 and -1.1 (data not shown here). In a condensed film (surface concentration of 0.45 µg/ cm2), the reorientation peak at approximately -0.8 V is visible and capacity peaks are shifted toward negative potential (Figure 8, curve 1) compared to the capacitance curve for DOMA monolayers of lower surface concentrations. Assuming that the surface of the lipid monolayer will change if NaPyS is present in the aqueous subphase, we did the following experiment. A NaPyS solution, concentration 4 µM, was placed in the electrochemical cell, and a DOMA monolayer was formed by spreading from hexane on the surface of the NaPyS solution and transferred to the mercury electrode by dipping the electrode through the layer. Capacity-potential curves of DOMA (surface concentration of 0.45 µg/cm2) on pure electrolyte (curve 1) and on 4 µM NaPyS (curve 2) are presented in Figure 8. The decrease of capacity and the shape of the (27) Lecompte, M. F.; Miller, I. R. Bioelectrochem. Bioenerg. 1979, 6, 537. (28) Pagano, R. E.; Miller, I. R. J. Colloid Interface Sci. 1973, 45, 126. (29) Zaba, B. N.; Wilkinson, M. C.; Taylor, D. M.; Lewis, T. J.; Laidman, D. L. FEBS Lett. 1987, 213, 49. (30) Nelson, A. J. Electroanal. Chem. 1995, 335, 327. (31) Miller, I. R.; Yavin, E. Bioelectrochem. Bioenerg. 1988, 19, 557. (32) Nelson, A.; Auffret, N.; Readman, J. Anal. Chim. 1988, 207, 47. (33) Nelson, A.; Leewen, van A. J. Electroanal. Chem. 1989, 273, 183.

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Kozarac et al. Table 2. Influence of DOMA Monolayers (0.45 µg/cm2) Spread on 0.5 M NaCl and on Solutions of NaPyS on Electrode Reduction of Cadmium conc of NaPyS (µM) 0 0.33 0.66 1.32 0.33 0.33 0.33 0.33 0.33

Figure 8. Capacity-potential curves for DOMA monolayers. Surface concentration: 0.45 µg/cm2. Subphase: (1) 0.55 M NaCl and (2) 0.55 M NaCl and 3 µM NaPyS.

tensametric curve for a DOMA monolayer on a NaPyS subphase changed compared to those for the layer on pure electrolyte, showing clearly the structural change of the DOMA monolayer in the presence of NaPyS in solution. Additional information on the adsorption behavior and structure of the adsorbed layer at the electrode surface can be obtained using the redox process of Cd(II) as a probe. Cadmium has been chosen because it has very convenient polarographic characteristics like a well defined, reversible, two-electron polarographic wave, extremely sensitive to the changes of the electrode reaction rate.34 It is known that lipid monolayers influence the electrode processes of cadmium by reducing the electrode reduction rate.10,33,35,36 The same was observed with DOMA monolayers.37 Our preliminary results on the adsorption processes of NaPyS on a bare mercury electrode showed that the reduction process of cadmium ions was not influenced even at a surface completely covered with the adsorbed layer on NaPyS.37 The influence of NaPyS adsorbed from the subphase on DOMA monolayers transferred to the mercury electrode can be seen from the (34) Tamamushi, R. Kinetic Parameters of Electrode Reactions of Metallic Compounds; Butterworths: London, 1975. (35) Jehring, H.; Huyen, N. V.; Gian, T. X.; Horn, E.; Hirche, C. J. Electroanal. Chem. 1979, 100, 13. (36) Kozarac, Z.; C Ä osovic´, B. Bioelectrochem. Bioenerg. 1984, 12, 353. (37) Kozarac, Z.; C Ä osovic´, B. In preparation.

accumulation time (min) 0 0 0 0 0 0.50 5 10 15

I/I0 0.21 0.21 0.75 0.88 0.21 0.30 0.75 0.80 0.90

results given in Table 2, where the dependence of the normalized peak height (I/I0) of the ac voltammograms of 10-5 M Cd2+ on the concentration of NaPyS in solution and on adsorption time is presented. In the ratio I/I0, the parameter I denotes the reduction current of 10-5 M Cd2+ across the DOMA monolayer, and the parameter I0 denotes the height of the ac voltammogram of Cd2+ obtained from pure electrolyte. It has been seen that, by increasing the concentration of NaPyS in the subphase solution and/or increasing adsorption time, the ratio I/I0 increased, showing enhanced transfer of cadmium through a DOMA monolayer modified by an adsorbed layer of NaPyS. 4. Conclusion 1. The preferential interaction of NaPyS with DOMA monolayers than with DPPC monolayers at the air/water interface was observed. This is due to the fact that the ionic interactions of the positively charged DOMA with PyS- anions are stronger than hydrogen bonding and hydrophobic interactions. 2. The enhanced transport of cadmium across the layers of DOMA and at the mercury electrode/NaPyS solution interface was observed and discussed in terms of the change in molecular organization of lipid films at the mercury surface in the presence of NaPyS in the bulk solution. Acknowledgment. We express our thanks to Werner Zeiss and Zvonimir Kodba for technical assitance. Financial support to Z.K. from the International Bureau of KFA, Ju¨lich, within the bilateral agreement between Germany and the Republic of Croatia is gratefully acknowledged. The work was funded by the Ministry of Science, Republic of Croatia, and the Bundesministerium fu¨r Forschung und Technologie. LA950908G